Comprehensive review of safety in Experimental Human Pneumococcal Challenge

Introduction Experimental Human Pneumococcal Challenge (EHPC) involves the controlled exposure of adults to a specific antibiotic-sensitive Streptococcus pneumoniae serotype, to induce nasopharyngeal colonisation for the purpose of vaccine research. The aims are to review comprehensively the safety profile of EHPC, explore the association between pneumococcal colonisation and frequency of safety review and describe the medical intervention required to undertake such studies. Methods A single-centre review of all EHPC studies performed 2011–2021. All recorded serious adverse events (SAE) in eligible studies are reported. An unblinded meta-analysis of collated anonymised individual patient data from eligible EHPC studies was undertaken to assess the association between experimental pneumococcal colonisation and the frequency of safety events following inoculation. Results In 1416 individuals (median age 21, IQR 20–25), 1663 experimental pneumococcal inoculations were performed. No pneumococcal-related SAE have occurred. 214 safety review events were identified with 182 (12.85%) participants presenting with symptoms potentially in keeping with pneumococcal infection, predominantly in pneumococcal colonised individuals (colonised = 96/658, non-colonised = 86/1005, OR 1.81 (95% CI 1.28–2.56, P = <0.001). The majority were mild (pneumococcal group = 72.7% [120/165 reported symptoms], non-pneumococcal = 86.7% [124/143 reported symptoms]). 1.6% (23/1416) required antibiotics for safety. Discussion No SAEs were identified directly relating to pneumococcal inoculation. Safety review for symptoms was infrequent but occurred more in experimentally colonised participants. Most symptoms were mild and resolved with conservative management. A small minority required antibiotics, notably those serotype 3 inoculated. Conclusion Outpatient human pneumococcal challenge can be conducted safely with appropriate levels of safety monitoring procedures in place.


Introduction
Experimental Human Pneumococcal Challenge (EHPC) involves the controlled exposure of adults to a specific antibiotic-sensitive Streptococcus pneumoniae serotype, to induce nasopharyngeal colonisation for the purpose of vaccine research. The aims are to review comprehensively the safety profile of EHPC, explore the association between pneumococcal colonisation and frequency of safety review and describe the medical intervention required to undertake such studies.

Methods
A single-centre review of all EHPC studies performed 2011-2021. All recorded serious adverse events (SAE) in eligible studies are reported. An unblinded meta-analysis of collated anonymised individual patient data from eligible EHPC studies was undertaken to assess the association between experimental pneumococcal colonisation and the frequency of safety events following inoculation.

Introduction
Controlled Human Infection Models (CHIM), in which individuals are exposed to pathogens of interest, provide a methodology for studying disease pathogenesis, understanding immunological correlates of protection, and assessing vaccine candidates' therapeutic efficacy. Currently, some regulators do not consider experimental challenge a medicinal product [1]; and therefore CHIMs do not necessarily have the same level of regulatory scrutiny as a Clinical Trial involving an Investigational Medicinal Product (CTIMP). However, the World Health Organisation (WHO) states that as these studies may evaluate the efficacy of future vaccines they should be treated similarly regarding regulations [2], in addition to Good Clinical Practice (GCP) compliance [3]. Given the risks inherent in replicating natural carriage/infection with a known pathogen, meticulous safety monitoring systems are crucial when protecting participants from harm.
In some CHIM studies participants are pre-emptively hospitalised for monitoring. This precaution is due to the challenge pathogen posing a potentially high level of risk to the participants' health if unmonitored and untreated, alongside the potential transmission risk to the public. Rigorous participant selection, education and early engagement with local health practitioners may mitigate this risk [4]. In other CHIMs, with asymptomatic colonisation, rather than disease as the aim, outpatient monitoring of well-informed participants may suffice.
The Experimental Human Pneumococcal Challenge (EHPC), a CHIM first developed in 2001 by McCool et al [5,6], has been used [7] to understand pneumococcal colonisation biology and links with transmission, correlates of protection and to accelerate pneumococcal vaccine development. Participants are experimentally inoculated with Streptococcus pneumoniae (pneumococcus), to explore immune responses mimicking "natural" exposure [7]. In this model, prevention of nasopharyngeal pneumococcal carriage acts as a surrogate for vaccineinduced immunity when testing vaccine efficacy [8], significantly reducing the required sample size and study duration. Previous research has demonstrated experimental colonisation with serotype 6B (SPN6B) to be asymptomatic in adults [9]; however other observational studies have reported mild symptoms in children during natural colonisation [10]. An increased frequency of upper respiratory tract (URT) symptoms was observed for serotype 3 [11], suggesting that serotype may impact the development of symptoms during colonisation.
CHIMs are considered safe for determining the efficacy of clinical interventions; however this has not been fully evidenced within pneumococcal CHIM through large-scale, objective analyses. Our aims are, therefore,: 1) A comprehensive description of all adverse events (AE, serious and non-serious) in EHPC at our centre 2) An assessment of AE severity (and hence re-consideration of the appropriateness of outpatient management) 3) An unblinded comparison of pneumococcal colonised/non-colonised experience of symptoms, including consideration of gender and other pneumococcal disease risk factors 4) Exploration of the influence of inoculum serotype on symptoms and 5) A review of safety processes and re-consideration of the level of precaution/effectiveness of interventions, to help inform other CHIM programmes.
We hypothesise that an outpatient pneumococcal challenge can be performed safely with appropriate monitoring, and that the pneumococcal colonisation status, challenge inoculation method and the administered serotype may influence the frequency of safety review required post-inoculation.

Methods
A single-centre review of safety data from EHPC studies at our centre between January 2011-June 2021, using an individual patient data meta-analysis approach for collated data. Inclusion criteria were: 1) Human participants 2) Outcome of nasopharyngeal pneumococcal colonisation, 3) Reported original results, and 4) Complete safety data available for review. Studies without extractable data were excluded. We accessed historical safety records and anonymised participant case report forms (CRFs) from published and unpublished studies. Data extraction was independently performed by 5 reviewers using a standardised data extraction form, with discrepancies resolved by group consensus.
All included studies had approval from a UK Research Ethics Committee (NHS Health Research Authority REC North West-Liverpool Central, REC IDs included in Table 1) and had been prospectively registered on a clinical trials database where required. Prospective approval for this service evaluation was obtained from the Liverpool University Hospitals Trust Audit Department (ID 10354). All participants provided written informed consent, and none had previously received a pneumococcal vaccine. Safety was defined as the absence of Serious Adverse Events (SAEs) directly related to experimental pneumococcal inoculation, as determined by an independent Data Safety and Monitoring Committee (DSMC).
An overview of EHPC study timepoints and procedures is illustrated in Fig 1A and the wider safety monitoring processes pre-and post-inoculation are summarised in Fig 1B. Inclusion and exclusion criteria utilised across all EHPC studies are detailed in S1 Table.

Participant symptom reporting and review
All participants were asked about the presence of symptoms at every visit, and if reported, underwent medical review by a study clinician. Symptomology, onset, severity, and duration were documented in the CRF. Further information on participant symptom assessment and investigation is detailed in the S1 File. Of note, study clinicians were unblinded to colonisation status throughout, impacting clinical suspicion during review. Participants were routinely blinded to their colonisation status until the end of follow-up.

Identification of participant episodes requiring safety review postexperimental challenge
All EHPC studies generate a weekly detailed safety report containing clinically relevant during the study period. Identified studies were reviewed in a two-stage process: 1. Study safety report review: Weekly safety reports for each study were reviewed for clinically relevant information on reported symptoms, extra clinical reviews, additional investigations, and all medically-significant occurrences. Potential clinical interactions were identified from these reports for further review.
2. Detailed CRF review: CRFs of relevant participants identified in the study safety report were reviewed by an experienced clinician to identify demographic information, study, challenge serotype and dose, inoculation method, colonisation status, and where safety incidents have occurred, alongside other key information pertaining to this.
Some EHPC studies involved multiple experimental challenges (with different serotypes and doses). Each challenge episode was treated as an individual event. All clinical episodes were reported separately unless deemed a continuation of the same symptom event. The follow-up period was considered closed at the last study visit. Data was collated in a purposebuilt Microsoft © Access database.

Defining SAE and Adverse Events (AE)
Definitions for key safety reporting terminology as defined in the Medicines and Healthcare products Regulatory Agency (MHRA) 'GCP guide' [3] were utilised, depending on the study protocol to standardise methods across CTIMP and non-CTIMP studies. These definitions were implemented as judged by consensus of an experienced clinical team and DSMC for SAE.

Defining potential pneumococcal related symptoms post-experimental challenge
S. pneumoniae is responsible for a broad spectrum of disease, meaning defining any presenting symptoms potentially related to pneumococcus is challenging. The UK Health Security Agency defines pneumococcal disease as 'invasive' and 'non-invasive', with five main clinical syndromes: meningitis, pneumonia, septicaemia (all invasive), otitis media and sinusitis (noninvasive) [18]. This study, therefore, focused on the symptomology of these disease areas. A literature review identified the clinical presentation and features of immunocompetent adults with these clinical syndromes due to pneumococcal infection (search terms detailed in S2 Table and results in S3 Table). This list was non-exhaustive, and clinician discretion was applied when assessing symptoms, with reviewer group consensus where a disagreement occurred. Medical events deemed non-related to pneumococcus were collected to ensure unexpected patterns of pneumococcal disease were not overlooked in the analysis.

Grading of participant symptom severity post-experimental challenge
No universally adopted system exists for grading AEs in CHIMs. To allow comparison the grading system (S4 Table, adapted from the Food and Drug Administration (FDA)/Center for Biologics Evaluation and Research, 'Grading of AE in healthy volunteers in vaccine trials' [19] was retrospectively applied by an independent clinician. If a participant received antibiotics or medical intervention, symptoms were deemed grade 2 or above.

Statistical analysis
Demographic data, colonisation data, and frequency of review were reported using descriptive statistics. The frequency, safety event type and symptoms in colonised and non-colonised individuals were compared using Fisher's Exact Test. An individual patient data meta-analysis methodology was utilised to estimate the association between pneumococcal colonisation and symptoms ('all reported' and 'potentially pneumococcal related') requiring safety review. Twostage meta-analysis using unadjusted binomial mixed effects models with a logit link and study ID as a random effect was used to estimate unadjusted associations (reported as odds ratio (OR), 95% confidence intervals [CI]) overall and by strata for visualisation in forest plots. Inverse variance weights and Hartung-Knapp adjustment were used for this model. While the interpretation of forest plots is limited as the data relates to a single centre, this method remains an effective means of visually displaying this data. A single-stage, co-variate adjusted, individual patient meta-analysis methodology was also utilised, again under a binomial mixed effects model with logit link, fitting the study as a random effect to identify an association with co-variates that may adjust or confound the association of interest. Sensitivity analyses were performed to assess the association with challenge serotype and inoculation method. All P-values were two-tailed and considered significant if P <0.05. All analyses were performed in R (version 4.2.0) or GraphPad Prism (version 9.20). (Table 1) were included with 1663 inoculations performed, involving 1416 participants (median age 21 [IQR 20-25], males = 568). Of these studies, 6 were RCTs (4 double-blinded, 2 partially-blinded) and 8 were interventional cohort studies. S1 Fig describes the study timelines. Two previously described inoculation methods were used: 1) 'Pipette-to-nose' technique [17] (100μl challenge inoculum directly pipetted into each nare, n = 1308) 2) 'Handto-nose' technique [14] (participant transfers challenge inoculum pipetted onto the dorsum of their hand to their nose under supervision, utilising a higher dose, n = 355). Across all studies pneumococcal colonisation was defined as the detection of experimental pneumococcus in nasal wash samples at any timepoint post-inoculation by classical microbiology, with latex agglutination testing for serotype confirmation.

eligible studies
Two studies were identified but not included in the full analysis: 1) 'Feasibility of Experimental Human Nasal Colonisation' (REC ID 08/H1001/52, n = 20) 2) 'Safety, Tolerability, and Efficacy Study of Prophylactic S. Pneumoniae Vaccine Following Challenge With S. Pneumoniae' [20] (REC ID 14/NW/0355, n = 100) as complete data was not obtainable. From the data available, no pneumococcal-related SAEs were reported in these studies.

No pneumococcal-related SAEs occurred post-experimental challenge
3 SAEs (3 individuals, 2 inoculated with SPN6B, 1 pre-inoculation, 0.21% of participants) were identified, all independently deemed unrelated to pneumococcal inoculation. All were significant medical events leading to hospitalisation and were considered SAEs. One participant (experimental SPN6B colonised) was admitted with tonsilitis for 48 hours. As non-toxigenic Corynebacterum diptheriae was cultured on throat swabs, this SAE was deemed unrelated to inoculation. The participant made a full, uneventful recovery following intravenous, then oral, antibiotics.
One further participant (experimental SPN3 colonised) reported an adverse event of special interest that was initially classed as an SAE. This individual developed otitis media with effusion following inoculation, progressing to tympanic perforation. A swab confirmed experimental SPN3 in aural discharge. They made a full recovery without sequelae, and following a detailed discussion with the DSMC this was downgraded to an AESI.

Safety review events occurred more frequently in pneumococcal colonised participants
Using the one-stage individual patient data meta-analysis approach the overall unadjusted association between pneumococcal colonisation and safety review (for any symptoms) was OR = 1.67 (95% CI 1.22-2.29). When adjusted to account for important co-variates (age, gender, inoculum, inoculum method) the OR is 1.49 (95% CI 1.08-2.06). The two-stage metaanalysis (covariate unadjusted) provides similar estimates (Fig 4), with a meta-analytic OR of 1.64 (95% CI 1.06-2.54). There is variation between studies, I 2 = 35% (P = 0.09), suggesting 35% of the heterogeneity in the association between pneumococcal colonisation and symptoms is attributable to inter-study heterogeneity.

Inoculum serotype influences symptomology with SPN3 inoculation associated with a higher frequency of reported safety events, grade of symptoms and requirement for antibiotics
Previous research has identified increased symptoms in those experimentally colonised with Spn3 [11] and this is driving the majority of the differences seen in colonised individuals. Forest plots from the two-stage meta-analysis (utilising a random effects model) with frequency of reported potential pneumococcal symptoms as outcome and stratified by inoculum serotype are presented (Fig 4B). The SPN3 group had a significantly higher OR for reported symptoms compared to SPN6B (SPN6B OR 1.68, 95% CI 1.10-2.56 vs SPN3 OR 6.45, 95% CI 2.43-17.11) and 23F (SPN23F OR 1.64, 95% CI 0.66-4.10).
Similarly, SPN3 colonisation influenced symptomology. When excluded from the analysis of the presenting symptoms (Fig 5B) only 'malaise' occurred more frequently in the colonised (colonised = 8/619, non-colonised = 2/948, P = 0.018, OR 6.17, 95% CI 1.53-28.97). The same was seen when symptoms leading to antibiotics were considered, with no significant difference observed for the remaining serotypes.
Another critical variable is the inoculation method. This variable had less influence on review frequency, despite the 'Hand-to-nose' method utilising a log 10 -higher inoculum dose for SPN6B. When the one-stage meta-analysis approach was stratified by inoculation method,  Table). OR were calculated using Fisher's Exact Test. A dotted line identifies an OR of 1 for comparison between symptoms. Only 'sore throat' and 'malaise' are statistically significant, with only 'malaise' significant when SPN3 inoculated individuals are removed. Three EHPC studies were CTIMPs (Table 1). Due to the increased regulatory requirements of CTIMPs, we hypothesised these would have a higher frequency of safety review episodes compared to non-CTIMPs. However, when adjusted for age, sex and inoculum received, CTIMPs had a similar OR for reporting pneumococcal-related symptoms as non-CTIMPs (CTIMPs OR 1.71, 95% CI 1.03-2.85, P = 0.038, Non-CTIMPs OR 2.02, 95% CI 1.28-3.19, P = 0.0003).

The frequency of non-pneumococcal symptoms was similar in pneumococcal colonised and non-colonised individuals
No significant difference in non-pneumococcal symptoms was observed between colonised and non-colonised (colonised 11/658 vs non-colonised 21/1005, P = 0.59, OR = 0.80, 95% CI 0.38-1.64). This held across all serotypes and inoculation methods. The type of safety review (at vs outside of routine appointment) was not significantly different. 7 participants commenced antibiotics for non-pneumococcal symptoms, with similar rates in colonised and noncolonised groups (colonised = 2/658, non-colonised = 5/1005, P = 0.71). The reported nonpneumococcal symptoms were diverse, with no identifiable symptomology trend.

Discussion
We have demonstrated that outpatient EHPC can be performed safely with appropriate screening and monitoring procedures as described. Symptoms requiring safety review are predominantly mild, infrequently require antibiotics, and their frequency is influenced by inoculum serotype. These data support the expanded use of the model to investigate additional serotypes, including adults at higher risk of pneumococcal disease (the primary target for improved pneumococcal vaccine development). Symptom review by research clinicians was influenced by knowledge of colonisation status, which likely affected clinical decision-making. Outpatient management is appropriate, but future risk assessments should consider inoculum serotype and maintain access to research-independent clinicians for colonisation-blinded review if severe symptoms develop. Fig 6 summarises the recommended features of a common EHPC protocol to ensure participant safety.
All three SAEs identified in this analysis were deemed unrelated to experimental inoculation. The frequency of symptoms requiring review was low, with symptom onset predominantly <7 days, supporting the continued use of an outpatient-based model. While there was variability in the frequency of reviews performed, only 12.92% of participants reported symptoms in keeping with pneumococcal disease. The majority were mild, only requiring assessment at routine appointments.
Low levels of heterogeneity were detected between studies, which was anticipated as all reported results are from one centre. However, the lack of a standardised severity grading measures limits the ability to compare the frequency and grade of adverse events occurring in individual CHIM programmes internationally. We suggest a standardised reporting methodology for all adverse events could allow wider comparisons between CHIM pathogens and ensure continued public confidence in CHIM methodology.
Pneumococcal colonised participants were reviewed more often than non-colonised (both for symptoms in keeping with pneumococcal disease and all-cause symptoms). This is likely due to the clinical team being aware of colonisation status during review, leading to extra vigilance for those colonised. We recommend blinding clinical staff to colonisation status where practical, ensuring all participants are managed objectively. However, the inoculum serotype utilised significantly impacts symptom frequency, with SPN3 associated with significantly more frequent and specific symptoms than other serotypes. When SPN3 is excluded, the remaining symptoms are predominantly mild, and antibiotic use is reducing substantially. This finding suggests SPN3 diverges from an asymptomatic colonisation model to one of controlled mild infection. While this is a significant change for EHPC, it is in keeping with other CHIM, such as the Group A Streptococcus [21] challenge, whose primary outcome is pharyngitis rather than colonisation. Future SPN3 studies should inform participants of the increased frequency and nature of these symptoms.
URT symptoms are frequently seen in respiratory viral infection, which was identified more often in colonised individuals, and can influence pneumococcal carriage [22,23]. As this analysis focused on safety, it is unclear from our data whether reported symptoms are occurring primarily due to pneumococcal colonisation, the development of infection or a symptomatic viral infection occurring (but not identified) co-incident with asymptomatic pneumococcal colonisation.
Identification of safety events is reliant on accurate record-keeping. As discussed there is potential for bias in reporting and management of symptomatic participants if colonisation status is known before review. The study is limited to a single centre and it is, therefore, unclear from the data whether these findings can be extrapolated to other centres. Two studies had incomplete safety records available. Retrospective symptom grading is subjective, even when performed by experienced medical teams. The medical team overseeing EHPC has evolved over the 10-year duration and there is likely to be some variability in clinical assessment. These limitations reduce the validity of conclusions regarding the frequency and type of symptoms occurring post-inoculation. However, this analysis provides a significant pooled population sample of a unique challenge methodology and applies robust statistical modelling. Importantly, the frequency of SAEs and overall antibiotic use is collated objectively across studies.

Conclusion
These data demonstrate an outpatient-based pneumococcal CHIM utilising varied challenge isolates, inoculation methods, and participants can be conducted safely with appropriate monitoring (common protocol described in Fig 6). Antibiotic use was low and safety reviews were infrequent, irrespective of colonisation status. SPN3 had a higher frequency and grade of symptoms, but this risk can be safely mitigated with close monitoring. Our data support the continued development of EHPC within higher-risk populations, and the development of similar outpatient challenge models for vaccine research. This study represents the experience of EHPC in a UK regulatory process and adjustments may be required when utilised elsewhere. This model has recently been transferred to a low-and-middle-income country (LMIC) [24], and these data suggest that an -adapted safety framework based on that described here, could also allow the transfer of an SPN3 model to this setting. Improved standardisation of adverse event reporting in CHIM would facilitate a direct comparison of safety between challenge pathogens. safety reviews across all included EHPC studies using a random effects model adjusting for inoculation method (pipette-tonose method or hand-to-nose method). IM = 1 refers to the' pipette-to-nose' method of inoculation and IM = 2 refers to the 'hand-to-nose method of inoculation. Effect sizes are reported as OR with 95% CI. The overall OR is shown in bold, and a dotted line included for comparison across studies. (DOCX) S1